Marker Of Cell Injury Research Articles

Correlation of serum chromium, zinc, magnesium and SOD levels with HbA1c in type 2 diabetes: A cross sectional analysis

The loss of dynamic integrity between homoeostasis of free radicals and antioxidants causes the development of complications like retinopathy, nephropathy, neuropathy, atherosclerosis and cardiovascular diseases in T2DM. Aims and ObjectivesTo assess the concentrations of serum chromium, zinc, magnesium and SOD in subjects of T2DM and control and to investigate the effect of these variables versus HbA1c. ResultsInsignificant difference (P=0.493) was reported in age (50±4.7 year compared with 50±7.2 year), while body mass Index (23±2kg/m2 compared with 26±4.5kg/m2) between the T2DM subjects and control subject showed significant difference (<0.0001). Inverse Pearson correlation coefficient, r (−0.376), (−0.689), (−0.05), (−0.05), (−0.40), (−0.14), (−0.342) and (−0.548) were established when HbA1c of control and T2DM patients were compared with control and T2DM patients of serum Cr, Zn, Mg and SOD variables in that order. The overall “p”-value demonstrated highly significant result at p<0.0001 between the T2DM subjects and controls. ConclusionStrong association between serum chromium and SOD in relation to HbA1c in this study gives a strong point that these variables could be used as markers of cell injury with the intention in further part of life en route to progressive complications in T2DM.

Xenon Blocks Neuronal Injury Associated with Decompression.

Despite state-of-the-art hyperbaric oxygen (HBO) treatment, about 30% of patients suffering neurologic decompression sickness (DCS) exhibit incomplete recovery. Since the mechanisms of neurologic DCS involve ischemic processes which result in excitotoxicity, it is likely that HBO in combination with an anti-excitotoxic treatment would improve the outcome in patients being treated for DCS. Therefore, in the present study, we investigated the effect of the noble gas xenon in an ex vivo model of neurologic DCS. Xenon has been shown to provide neuroprotection in multiple models of acute ischemic insults. Fast decompression compared to slow decompression induced an increase in lactate dehydrogenase (LDH), a well-known marker of sub-lethal cell injury. Post-decompression administration of xenon blocked the increase in LDH release induced by fast decompression. These data suggest that xenon could be an efficient additional treatment to HBO for the treatment of neurologic DCS.

Invited Commentary

Invited Commentary

Diagnostic Value of Serum Levels of GFAP, pNF-H, and NSE Compared With Clinical Findings in Severity Assessment of Human Traumatic Spinal Cord Injury.

An analytical cohort study. This study aimed to evaluate severity of traumatic spinal cord injury (SCI) based on the serum levels of phosphorylated form of heavy subunit of neurofilament (pNF-H), neuron-specific enolase (NSE), and glial fibrillary acidic protein (GFAP), which are axonal, neural cell body, and glial cell injury markers, respectively. Prior studies have reported elevated serum levels of pNF-H, NSE, and GFAP as biomarkers for the detection of traumatic SCI in animals. However, in this study, these biomarkers were studied in humans and with an extended level of timing. The study included 35 patients with SCI with a mean age of 36.5 years. All patients were evaluated using the American Spinal Injury Association Impairment Scale, followed by examinations including radiography and spinal computed tomography for determining the injury level. Serum levels of NSE, pNF-H, and GFAP were determined using enzyme-linked immunosorbent assay. The mean serum level of GFAP was significantly higher in patients with SCI than in the control group. Mean serum levels of pNF-H and NSE were significantly higher during 24 and 48 hours after injury in patients with SCI than in the control group. The serum level of GFAP was appropriate for estimating the severity of SCI in the first 24 hours after injury. Our findings suggest that increased serum levels of GFAP, NSE, and pNF-H can be used for the diagnosis and degree of SCI severity in trauma patients. During 48 hours after injury, estimation of serum levels of pNF-H, NSE, and GFAP, combined with neurological testing, could predict the presence of SCI and severity prior to spinal computed tomography and surgical or conservative interventions. 2.

Dysfunction of liver regeneration in aged liver after partial hepatectomy.

A remarkable feature of the liver is its regenerative capacity following partial hepatectomy. However, the regenerative capacity of many organs and tissues loses its natural ability to divide with aging. In this study, we investigated the association of aging with endoplasmic reticulum stress, the cell cycle, autophagy, and apoptosis-related genes during liver regeneration after hepatectomy. Balb/c 4-week and 40-week-old male mice were subjected to 70% hepatectomy. Animals were sacrificed at 24, 48, and 72 h after hepatectomy. Immunohistochemical stainings for proliferating cell nuclear antigen, LC3, Atg5, and caspase-3 were used to quantify protein expression. Real-time reverse transcription-polymerase chain reaction was used to detect p16, CHOP, LC3, Atg5, hepatocyte growth factor, cMet, cyclin D1, cyclin A2, and caspase-3 expression. After hepatectomy, old group showed a lower survival rate and significantly lower expression of hepatocyte growth factor, cMet, cyclin D1, cyclin A2, proliferating cell nuclear antigen labeling index, and SMP30 compared with young group. The liver weight/body weight ratio was significantly lower at 48 h and 72 h after hepatectomy and was accompanied by markedly elevated levels of the liver cell injury markers, LC3 and caspase-3. Immunohistochemical results showed that LC3, Atg5, and caspase-3 protein expression were higher in old group than in young group. These results revealed that impaired liver regeneration was due to aging, which was expressed by decreased cell cycle and increased autophagy and apoptosis. Therefore, understanding the molecular basis for aged liver regeneration might provide a new therapeutic option for old patients.

SIRT1 reduction causes renal and retinal injury in diabetes through endothelin 1 and transforming growth factor β1

In diabetes, hyperglycaemia causes up-regulation of endothelin 1 (ET-1) and transforming growth factor beta 1 (TGF-β1). Previously we showed glucose reduces sirtuin1 (SIRT1), a class III histone deacetylase. Here, we investigated the regulatory role of SIRT1 on ET-1 and TGF-β1 expression. Human microvascular endothelial cells were examined following incubation with 25 mmol/l glucose (HG) and 5 mmol/l glucose (NG) with or without SIRT1 or histone acetylase p300 overexpression or knockdown. mRNA expressions of ET-1, TGF-β1, SIRT1, p300 and collagen 1α(I) were examined. SIRT1 enzyme activity, ET-1 and TGF-β1 protein levels were measured. Histone acetylation and endothelial permeability were further investigated. Similar analyses were performed in the kidneys and retinas of SIRT1 overexpressing transgenic mice with or without streptozotocin induced diabetes. Renal functions were evaluated. In the endothelial cells (ECs), HG caused increased permeability and escalated production of ET-1, TGF-β1, collagen Iα(I). These cells also showed increased p300 expression, histone acetylation and reduced SIRT1 levels. These changes were rectified in the ECs following p300 silencing or by SIRT1 overexpression, whereas SIRT1 knockdown or p300 overexpression in NG mimicked the effects of HG. High ET-1 and TGF-β1 levels were seen in the kidneys and retinas of diabetic mice along with micro-albuminuria and increased fibronectin protein (marker of glucose-induced cell injury) levels. Interestingly, these detrimental changes were blunted in SIRT1 overexpressing transgenic mice with diabetes. This study showed a novel SIRT1 mediated protection against renal and retinal injury in diabetes, regulated through p300, ET-1 and TGF-β1.

Immunological response and markers of cell damage in seropositive horses for Toxoplasma gondii.

Toxoplasmosis is an important parasitic disease affecting several species of mammals, but little is known about this disease in horses. This study aimed to investigate the levels of several immunological variables and markers of cell damage in the serum of seropositive horses for Toxoplasma gondii. Sera samples of adult horses from the Santa Catarina State, Brazil used on a previous study were divided into groups according to their antibody levels for T. gondii determined by immunofluorescence assay, i.e. 20 samples from seronegative horses (Group A - control), 20 samples from horses with titers of 1:64 (Group B), 20 samples of horses with titers of 1:256 (Group C), and five samples from horses with titers of 1:1024 (Group D). Positive animals (Groups B, C, and D) had higher levels of immunoglobulins (IgM and IgG), pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1, IL-4, and IL-6) and protein C-reactive protein, as well as lower levels of IL-10 (anti-inflammatory cytokine) when compared to seronegative horses (Group A). The nitric oxide levels were also elevated in seropositive horses. Therefore, we have found humoral and cellular immune responses in seropositive horses, and a correlation between high antibody levels and inflammatory mediators. Markers of cell injury by lipid peroxidation (TBARS) and protein oxidation (AOPP) were elevated in animals seropositives for T. gondii when compared to seronegatives. Therefore, seropositive horses to T. gondii can keep active immune responses against the parasite. As a consequence with chronicity of disease, they show cellular lesions that may lead to tissue damage with the appearance of clinical disease.

Abstract 20538: Nrf2 Signaling Ameliorates Ventilator-Induced Lung Inflammation

Ventilator-induced lung injury (VILI) is an acute lung injury that develops during mechanical ventilation with high tidal volume in patients in the intensive care unit (ICU). VILI is a common entity with an unacceptable mortality rate (~20%). As no current therapies exist for VILI, novel therapeutic targets for VILI and mechanical stress-induced tissue injury are needed. In this study, we defined Nrf2 signaling in VILI and mechanical stress-induced endothelial cell pathobiology. Nrf2 is a transcription factor that alters ROS balance and is a novel therapeutic target for VILI. First, we defined Nrf2 as a sensory marker for mechanical stress-induced endothelial cell injury. Antioxidant response element (ARE) luciferase plasmids were transfected to both endothelial cells and murine lung tissues. EC exposed to 18% cyclic stretch (18% CS) or mice exposed to excessive tidal volume ventilation (40 ml/kg) resulted in ARE activation, site of Nrf2 binding. We next confirmed Nrf2 signaling is essential for gene expression dysregulation by 18% CS via Nrf2 knockdown that significantly ameliorates 18% CS-induced gene expression pattern, including signaling pathways regulating endothelial barrier function including MYLK. Finally, we validated that Nrf2 is a novel and effective therapeutic target for VILI in pre-clinical models of VILI. Nrf2 activator sulforaphane (SF, 11.5 mg/kg) significantly attenuated VILI (40 ml/kg, 4 hrs)-induced lung inflammation, including BAL protein leakage and white blood cell infiltration. These novel findings characterized Nrf2 as a sensor for mechanical stress, a central mediator of mechanical stress induced gene expression dysregulation, and a novel therapeutic target for VILI.

Severity of lethal ischemia/reperfusion injury in rat hearts subjected to ischemic preconditioning is increased under conditions of simulated hyperglycemia.

The aim of our study was to characterize resistance to ischemia/reperfusion (I/R) injury in Langendorff-perfused rat hearts and effectivity of ischemic preconditioning (PC) under condition of simulated acute hyperglycemia (SAHG) by perfusion of the hearts with Krebs-Henseleit (KH) solution with elevated glucose concentration (22 mmol/l). I/R injury was induced by 30-min coronary occlusion followed by 120-min reperfusion and PC by two cycles of 5-min occlusion/5-min reperfusion, prior to I/R. The severity of I/R injury was characterized by determination of the size of infarction (IS, expressed in % of area at risk size) and the amount of heart-type fatty acid binding protein (h-FABP, a marker of cell injury) released from the hearts to the effluent. Significantly smaller IS (8.8+/-1 %) and lower total amount of released h-FABP (1808+/-660 pmol) in PC group compared with IS 17.1+/-1.2 % (p<0.01) and amount of h-FABP (8803+/-2415 pmol, p<0.05) in the non-PC control hearts perfused with standard KH solution (glucose 11 mmol/l) confirmed protective effects of PC. In contrast, in SAHG groups, PC enhanced IS (21.4+/-2.2 vs. 14.3+/-1.3 %, p<0.05) and increased total amount of h-FABP (5541+/-229 vs. 3458+/-283 pmol, p<0.05) compared with respective non-PC controls. Results suggest that PC has negative effect on resistance of the hearts to I/R injury under conditions of elevated glucose in vitro.

YKL-40, a Marker of Cardiovascular Disease and Endothelial Dysfunction, in Kidney Transplant Recipients

BackgroundYKL-40 is an inflammatory glycoprotein involved in endothelial dysfunction and expressed in macrophages in the earliest lesion of atherosclerosis. Because cardiovascular mortality is the main cause of death, there are no data on kidney transplant recipients, so the aim of the study was to assess YKL-40 in that population, with particular attention being paid to the relationship with endothelial damage. MethodsWe studied 68 patients after kidney transplantation. Complete blood count, creatinine, lipids, and fasting glucose were studied with the use of standard laboratory methods. We assessed YKL-40; markers of inflammation high-sensitivity C-reactive protein (hs-CRP), von Willebrand factor, thrombomodulin, interleukin-6, intracellular adhesion molecule, and vascular cell adhesion molecule; markers of hemostasis plasmin-antiplasmin complexes, thrombin-antithrombin complexes, prothrombin fragments 1+2; and marker of kidney function neutrophil gelatinase–associated lipocalin (NGAL) with the use of commercially available assays. ResultsMean levels of YKL-40 were significantly higher in kidney allograft recipients than in the control group (P < .001). YKL-40 was also significantly higher in patients with coronary artery disease, hypertension, and diabetes compared with their counterparts without these diseases. YKL-40, in univariate analysis, was related to NGAL, protein Z, plasmin-antiplasmin complex, mean corpuscular volume, cyclosporine level, and hs-CRP. YKL-40 was not related to serum lipids, markers of endothelial cell injury, or causes of end-stage renal failure. YKL-40 was predicted by cyclosporine level, NGAL, and hs-CRP, explaining 37% of the variation. ConclusionsElevated YKL-40 may contribute to enhanced risk of cardiovascular complication, mainly owing to endothelial cell injury and inflammation in patients after kidney transplantation treated with calcineurin inhibitors.

Inhibiting Monoacylglycerol Acyltransferase 1 Ameliorates Hepatic Metabolic Abnormalities but Not Inflammation and Injury in Mice

Abnormalities in hepatic lipid metabolism and insulin action are believed to play a critical role in the etiology of nonalcoholic steatohepatitis. Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol, which is the penultimate step in one pathway for triacylglycerol synthesis. Hepatic expression of Mogat1, which encodes an MGAT enzyme, is increased in the livers of mice with hepatic steatosis, and knocking down Mogat1 improves glucose metabolism and hepatic insulin signaling, but whether increased MGAT activity plays a role in the etiology of nonalcoholic steatohepatitis is unclear. To examine this issue, mice were placed on a diet containing high levels of trans fatty acids, fructose, and cholesterol (HTF-C diet) or a low fat control diet for 4 weeks. Mice were injected with antisense oligonucleotides (ASOs) to knockdown Mogat1 or a scrambled ASO control for 12 weeks while remaining on diet. The HTF-C diet caused glucose intolerance, hepatic steatosis, and induced hepatic gene expression markers of inflammation, macrophage infiltration, and stellate cell activation. Mogat1 ASO treatment, which suppressed Mogat1 expression in liver and adipose tissue, attenuated weight gain, improved glucose tolerance, improved hepatic insulin signaling, and decreased hepatic triacylglycerol content compared with control ASO-treated mice on HTF-C chow. However, Mogat1 ASO treatment did not reduce hepatic diacylglycerol, cholesterol, or free fatty acid content; improve histologic measures of liver injury; or reduce expression of markers of stellate cell activation, liver inflammation, and injury. In conclusion, inhibition of hepatic Mogat1 in HTF-C diet-fed mice improves hepatic metabolic abnormalities without attenuating liver inflammation and injury.

Cost-efficient method and device for the study of stationary tissular gas bubble formation in the mechanisms of decompression sickness.

Current in vivo methods cannot distinguish between the roles of vascular and stationary tissular gas bubbles in the mechanisms of decompression sickness (DCS). To answer this question, we designed a normobaric-hyperbaric chamber for studying specifically the contribution of stationary tissular gas bubbles in the mechanisms of DCS in individually-superfused tissue samples. For validating our method, we investigated in rat brain slices exposed to 0.4MPa air absolute pressure whether fast decompression rate - the most important cause of cerebral DCS - may induce an increase of lactate dehydrogenase (LDH), a marker of cell injury, compared to slow decompression rate. We provide a technical description of our pressure chamber and show that fast decompression rate of 0.3MPamin(-1) induced a rapid and sustained increase of LDH release compared to slow compression rate of 0.01MPamin(-1) (P<0.0001). There is no current method for studying stationary tissular gas bubbles. This report describes the first method for studying specifically in tissue samples the role of stationary tissular gas bubbles in the mechanisms of DCS. Advantageously, according to this method (i) biological markers other than LDH could be easily studied; (ii) tissue samples could be taken not only from the brain but also from any part of the animal's body known of interest in DCS research, allowing performing tissue compartment research, a major question in the physics and theory of decompression research; and (iii) histological studies could be performed from the tissue samples.

Oxalate at physiological urine concentrations induces oxidative injury in renal epithelial cells: effect of α‐tocopherol and ascorbic acid

To test our hypothesis that physiological levels of urinary oxalate induce oxidative renal cell injury, as studies to date have shown that oxalate causes oxidative injury only at supra-physiological levels. To study the combined effect of α-tocopherol and ascorbic acid against oxalate-induced oxidative injury, as oxalate-induced oxidative cell injury is known to promote initial attachment of calcium oxalate crystals to injured renal tubules and subsequent development of kidney stones. Cultures of normal (antioxidant-undepleted) and antioxidant-depleted LLC-PK1 cells were exposed to oxalate at human physiological urine concentrations. After exposure, markers of oxidative stress and cell injury were measured in the cells and media, respectively. In addition, we also evaluated the combined effects of α-tocopherol and ascorbic acid on oxalate-induced oxidative cell injury. Exposure of renal cells to oxalate at urinary physiological levels increased the oxidative cell injury as assessed by increased lactate dehydrogenase (LDH) leakage and increased lipid hydroperoxide in the renal cells; however, this effect was not seen until 24 h after oxalate exposure, at which point the injury was milder. On the other hand, when cellular reduced glutathione (GSH) and catalase were depleted in renal epithelial cells with pharmacological inhibitors, the physiological levels of urinary oxalate caused significant oxidative cell injury at 24 h, and remarkably, when additional endogenous antioxidants were depleted, the oxalate at the upper limit of normal 24 h urine caused a significant amount of cell injury in a shorter period of time, which was comparable to that seen in cells exposed to higher levels of oxalate. Exposure of LLC-PK1 cells to oxalate resulted in increased levels of H2 O2 and lipid hydroperoxide, correlating with increased release of cell injury markers, including LDH, alkaline phosphate, and γ-glutamyl transpeptidase from renal tubular epithelial cells. Oxalate exposure decreased the activity and protein expression of superoxide dismutase and glutathione peroxidase in a time-dependent manner. LLC-PK1 cells treated with oxalate and either α-tocopherol or ascorbic acid alone exhibited a significant decrease in oxidative cell injury and restored endogenous renal antioxidants towards normal levels, and interestingly, combined treatment with α-tocopherol and ascorbic was more efficient at preventing oxalate-induced toxicity than treatment with either agent alone. To our knowledge this is the first study to show that oxalate alone at human physiological urine concentrations (in the absence of calcium oxalate crystal formation), induced oxidative renal injury in renal epithelial cells when endogenous antioxidants are depleted. Our data further suggests that a combination of α-tocopherol and ascorbic acid may be more effective than each individual agent in reducing oxalate-induced oxidative renal injury and subsequent calcium oxalate crystal deposition in recurrent stone formers.

Assessment of biochemical liver markers, physical activity, fitness and body mass index for a cardiometabolic risk model in childhood

The aim of this study was to investigate clustered cardiometabolic risk scores in healthy 10- to 12-year-olds using anthropometric characteristics, measurements of cardiorespiratory fitness (CRF) and physical activity and blood markers of metabolic disease. We also evaluated how including markers of liver cell injury would affect the clustered cardiometabolic risk assessment model. This cross-sectional study focused on 99 children aged 10-12 years. The main outcome included assessing participants with increased and low cardiometabolic risk factors using a clustered risk score model that incorporated markers implicated in metabolic syndrome pathogenesis. Two clustered risk scores were calculated, one incorporating markers of liver cell injury. Children classified as 'increased risk' exhibited significantly lower CRF and higher body mass index Z-scores than their 'low-risk' peers. No significant differences in physical activity were observed. This trend remained unchanged when markers of liver injury were included in the clustered risk assessment model. The clustered risk score model is a scientifically robust method of cardiometabolic risk assessment, which reiterates the importance of weight reduction and CRF promotion in childhood. Our study did not show a significant contribution of liver injury markers, and further research is needed to evaluate their effect on cardiometabolic risk stratification in childhood.

Nitrosonifedipine Ameliorates the Progression of Type 2 Diabetic Nephropathy by Exerting Antioxidative Effects

Diabetic nephropathy (DN) is the major cause of end-stage renal failure. Oxidative stress is implicated in the pathogenesis of DN. Nitrosonifedipine (NO-NIF) is a weak calcium channel blocker that is converted from nifedipine under light exposure. Recently, we reported that NO-NIF has potential as a novel antioxidant with radical scavenging abilities and has the capacity to treat vascular dysfunction by exerting an endothelial protective effect. In the present study, we extended these findings by evaluating the efficacy of NO-NIF against DN and by clarifying the mechanisms of its antioxidative effect. In a model of type 2 DN (established in KKAy mice), NO-NIF administration reduced albuminuria and proteinuria as well as glomerular expansion without affecting glucose metabolism or systolic blood pressure. NO-NIF also suppressed renal and systemic oxidative stress and decreased the expression of intercellular adhesion molecule (ICAM)-1, a marker of endothelial cell injury, in the glomeruli of the KKAy mice. Similarly, NO-NIF reduced albuminuria, oxidative stress, and ICAM-1 expression in endothelial nitric oxide synthase (eNOS) knockout mice. Moreover, NO-NIF suppressed urinary angiotensinogen (AGT) excretion and intrarenal AGT protein expression in proximal tubular cells in the KKAy mice. On the other hand, hyperglycemia-induced mitochondrial superoxide production was not attenuated by NO-NIF in cultured endothelial cells. These findings suggest that NO-NIF prevents the progression of type 2 DN associated with endothelial dysfunction through selective antioxidative effects.

Role of Pulsatility in Hypothermic Reconditioning of Porcine Kidney Grafts by Machine Perfusion After Cold Storage

Brief in-house machine perfusion after cold storage (CS) (hypothermic reconditioning) has been proposed as a convenient tool to improve kidney graft function. The present study aimed to investigate the mechanistic role of vascular pulsatility in this context. Kidney function after cold preservation (4°C, 18 hr) and subsequent reconditioning by 90 min of pulsatile machine perfusion (PP) (30/20 mm Hg) or nonpulsatile machine perfusion (NPP) (30 mm Hg) was studied in an isolated kidney perfusion model in pigs (n=6 for both) and compared with simply CS grafts. Compared with CS, PP but not NPP significantly improved renal perfusate flow and urine production and significantly increased the reduction of perfusate levels of creatinine and urea during reperfusion. Perfusate levels of fatty acid binding protein, a marker of tubular cell injury, were dramatically reduced by PP but not NPP. PP and NPP lowered fractional excretion of sodium, but significance was only reached for PP. Molecular effects of PP comprised a significant (vs. CS) mRNA elevation of the endothelial anti-inflammatory transcription factor Krüppel-like factor 2 as well as endothelial nitric oxide synthase, along with significantly higher perfusate levels of the endogenous vasodilator nitric oxide. Functional efficiency of PP over CS was confirmed in additional porcine transplant experiments (n=5 for both) by, for example, up to threefold improved clearance of creatinine during the first days after transplantation. PP of 90 min shortly before transplantation seems to be an efficient mechanism to reduce proinflammatory endothelial phenotype and improve functional outcome of kidney grafts even after preceding static storage.

Quiescent complement in nonhuman primates during E coli Shiga toxin-induced hemolytic uremic syndrome and thrombotic microangiopathy

AbstractEnterohemorrhagic Escherichia coli (EHEC) produce ribosome-inactivating Shiga toxins (Stx1, Stx2) responsible for development of hemolytic uremic syndrome (HUS) and acute kidney injury (AKI). Some patients show complement activation during EHEC infection, raising the possibility of therapeutic targeting of complement for relief. Our juvenile nonhuman primate (Papio baboons) models of endotoxin-free Stx challenge exhibit full spectrum HUS, including thrombocytopenia, hemolytic anemia, and AKI with glomerular thrombotic microangiopathy. There were no significant increases in soluble terminal complement complex (C5b-9) levels after challenge with lethal Stx1 (n = 6) or Stx2 (n = 5) in plasma samples from T0 to euthanasia at 49.5 to 128 hours post-challenge. d-dimer and cell injury markers (HMGB1, histones) confirmed coagulopathy and cell injury. Thus, complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these preclinical models, and benefits or risks of complement inhibition should be studied further for this infection.

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

Cryptococcus neoformans promotes its transmigration into the central nervous system by inducing molecular and cellular changes in brain endothelial cells.

Cryptococcus spp. cause fungal meningitis, a life-threatening infection that occurs predominately in immunocompromised individuals. In order for Cryptococcus neoformans to invade the central nervous system (CNS), it must first penetrate the brain endothelium, also known as the blood-brain barrier (BBB). Despite the importance of the interrelation between C. neoformans and the brain endothelium in establishing CNS infection, very little is known about this microenvironment. Here we sought to resolve the cellular and molecular basis that defines the fungal-BBB interface during cryptococcal attachment to, and internalization by, the human brain endothelium. In order to accomplish this by a systems-wide approach, the proteomic profile of human brain endothelial cells challenged with C. neoformans was resolved using a label-free differential quantitative mass spectrometry method known as spectral counting (SC). Here, we demonstrate that as brain endothelial cells associate with, and internalize, cryptococci, they upregulate the expression of several proteins involved with cytoskeleton, metabolism, signaling, and inflammation, suggesting that they are actively signaling and undergoing cytoskeleton remodeling via annexin A2, S100A10, transgelin, and myosin. Transmission electronic microscopy (TEM) analysis demonstrates dramatic structural changes in nuclei, mitochondria, the endoplasmic reticulum (ER), and the plasma membrane that are indicative of cell stress and cell damage. The translocation of HMGB1, a marker of cell injury, the downregulation of proteins that function in transcription, energy production, protein processing, and the upregulation of cyclophilin A further support the notion that C. neoformans elicits changes in brain endothelial cells that facilitate the migration of cryptococci across the BBB and ultimately induce endothelial cell necrosis.

Plasma Levels of sRAGE, Loss of Aeration and Weaning Failure in ICU Patients: A Prospective Observational Multicenter Study

RationalePostextubation distress after a successful spontaneous breathing trial (SBT) is associated with increased morbidity and mortality. Lung ultrasound determination of changes in lung aeration predicts weaning failure. It remains unknown whether this derecruitment is related to alveolar epithelial dysfunction or not.ObjectiveTo verify whether lung alveolar type I epithelial cell injury marker sRAGE (soluble form of the receptor for advanced glycation end-products) is predictive of postextubation distress and weaning failure or not, and to verify whether plasma sRAGE levels can be related to lung derecruitment during the process of weaning from mechanical ventilation or not.Interventions, Measurements88 patients from 2 intensive care units were included in this observational prospective study. Plasma sRAGE levels were measured in duplicate by ELISA before, at the end of a 60-minute SBT, and 4 hours after extubation. To quantify lung aeration, a lung ultrasound score was calculated.Main Results34% of extubated patients experienced postextubation distress. Patients with or without postextubation distress had comparable sRAGE levels before SBT, after SBT, and 4 hours after extubation. In patients with postextubation distress, sRAGE levels were not predictive of the need for mechanical ventilation. sRAGE levels were not associated with lung aeration as assessed by echography. Patients who succeeded SBT (86%) and those who failed (14%) had no differences in sRAGE levels, before (median 1111 vs 1021 pg/mL, p = 0,87) and at the end of SBT (1165 vs 1038 pg/mL, p = 0.74).ConclusionsPlasma levels of sRAGE do not predict postextubation distress or SBT failure/success in patients weaning from mechanical ventilation. Lung aeration loss during a successful weaning trial predicts postextubation distress, but may not be evaluable by plasma levels of sRAGE, a marker of alveolar type I epithelial cell injury.Trial RegistrationClinicalTrials.gov NCT01098773